Cyclophosphamide-loaded drug delivery system based on PAMAM (Polyamidoamine) dendrimer as well as preparation method and application of cyclophosphamide-loaded drug delivery system

Abstract

The invention discloses a PAMAM (Polyamidoamine) dendrimer-based cyclophosphamide-loaded drug delivery system and a preparation method and application thereof, the antioxidant characteristic of polyphenol and various advantages of PAMAM are combined, bovine serum albumin is introduced to reduce the cytotoxicity of high-algebraic PAMAM strong cations, and loaded cyclophosphamide targets a diseased region, so that the drug delivery system has a good application prospect. The effects of resisting oxidation, resisting inflammation, regulating immunity and the like are effectively exerted. The prepared nanogel delivery system has good biocompatibility, can target an extracellular matrix with negative charges and efficiently remove active oxygen so as to effectively solve the problem that cyclophosphamide is lack of specificity in in-vivo distribution and the like, the bioavailability of cyclophosphamide is improved, the nanogel delivery system is applied to treatment of systemic sclerosis for the first time, and the nanogel delivery system has broad application prospects. And a promising treatment strategy is provided for the research of fibrosis diseases.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to a drug delivery system based on PAMAM dendrimer macromolecules carrying cyclophosphamide, and a preparation method and application thereof. Background Art

[0002] Systemic sclerosis (SSc), also known as scleroderma, is a chronic autoimmune disease characterized by microvascular damage, immune dysfunction and multi-organ fibrosis. The continuous progression of vascular damage and fibrosis makes SSc the autoimmune disease with the highest mortality rate. Current treatment focuses on symptomatic treatment and prevention of complications, and there is no specific drug that can completely cure the disease. Immunosuppressants and immunomodulatory therapy are the core of SSc treatment, with the goal of rebuilding the immune system. Cyclophosphamide, as an effective immunosuppressant, is widely used in clinical practice. It can slow down the progression of skin and lung fibrosis and significantly improve the quality of life of patients. Cyclophosphamide (CYC) is soluble in water, but its solubility is not high, and its aqueous solution is unstable, so it should be used within a short period of time after dissolution. However, traditional drug administration methods such as direct intravenous injection will cause the drug to enter the systemic blood circulation, and only a small amount will reach the lesion site, with large side effects. Micro-scale carriers such as nanohydrogel materials are expected to overcome the above shortcomings, but these materials still have some limitations, such as poor injectability, biocompatibility, and low cell uptake rate. Therefore, there is an urgent need to find new strategies that can improve cellular uptake in lesion sites for the encapsulation and delivery of cyclophosphamide, so that it can be targeted to the lesion site, enhance the therapeutic effect of cyclophosphamide, and improve the overall immune regulation ability. Summary of the invention

[0003] Purpose of the invention: The technical problem to be solved by the present invention is to provide a nanogel-loaded cyclophosphamide delivery system formed by combining catechol-modified PAMAM with bovine serum albumin to improve the drug delivery efficiency. At the same time, combined with the excellent antioxidant capacity of catechol, active oxygen is scavenged, oxidative stress is targeted, and systemic sclerosis is more effectively treated.

[0004] In order to achieve the above object, the technical solution adopted by the present invention is as follows: A method for preparing a cyclophosphamide drug delivery system based on PAMAM dendrimers comprises the following steps: (1) Synthesis of polyphenol-modified PAMAM 3,4-dihydroxybenzaldehyde and G5 PAMAM were used as raw materials for reaction and then reduced with sodium cyanoborohydride to generate polyphenol-modified PAMAM, referred to as PDP; (2) Preparation of PDP@BSA solution The PDP synthesized in step (1) was mixed with BSA and incubated at room temperature for 20-40 minutes, centrifuged to obtain PDP@BSA, and redispersed in deionized water; (3) Loading cyclophosphamide The cyclophosphamide aqueous solution is added dropwise to the PDP@BSA solution synthesized in step (2) for coating, and the mixture is obtained by centrifugation.

[0005] Among them, polyamidoamine (PAMAM) dendrimers are a type of dendrimer material that is currently widely used and is known as "artificial protein". The PAMAM dendrimer structure is precisely controllable, and not only can small molecular weight drugs be loaded into its internal hydrophobic cavity structure, but also stimulus-responsive drugs can be connected to the surface through covalent bonds, and can be used as a carrier for immunomodulatory therapy.

[0006] Natural polyphenols play an important role in cell protection, metal coordination and signal transduction due to their various biological activities, such as antioxidant, anticancer, antibacterial and antiviral. Natural polyphenols can also combine with various substances to form nanoparticles through hydrogen bonds, hydrophobic interactions, π-π interactions and cation-π interactions, which have broad application prospects in drug delivery and disease treatment. They can improve the stability and bioavailability of drugs and improve the delivery efficiency of drugs.

[0007] Human serum albumin (HSA) is the most abundant protein in human plasma and has multiple physiological functions, including maintaining blood osmotic pressure and transporting a variety of small molecules. Bovine serum albumin (BSA) and HSA have a high degree of similarity in amino acid sequence, which, together with the advantages of high purity and low cost, makes it an ideal carrier for the preparation of biological nanoparticles.

[0008] The present invention is based on the typical glycopeptide composition structure of the extracellular matrix (ECM), combines polyphenol-modified PAMAM dendrimers and bovine serum albumin together to encapsulate cyclophosphamide, and obtains a PDP@BSA / CYC complex, which has a good effect in treating systemic sclerosis.

[0009] Preferably, in step (1), the G5 PAMAM is a fifth generation polyamide-amine dendrimer, whose surface groups are amino groups and whose molecular weight is 28826.

[0010] Preferably, in step (1), the molar ratio of the 3,4-dihydroxybenzaldehyde to the G5 PAMAM is (24:1) to (128:1); the molar ratio of the sodium cyanoborohydride to the 3,4-dihydroxybenzaldehyde is (1.5:1) to (3:1).

[0011] Specifically, in step (1), 3,4-dihydroxybenzaldehyde and G5 PAMAM are dissolved in an anhydrous methanol solution, mixed and reacted at 2-8°C for 20-40 hours, and anhydrous acetic acid is added as a catalyst; sodium cyanoborohydride is then added to continue the reaction for 4-8 hours, and after the reaction, the product is fully dialyzed with deionized water to remove impurities, and finally the purified product is collected by vacuum freeze drying to obtain PDP.

[0012] Preferably, in step (2), the number average molecular weight of BSA is about 66-67 kDa.

[0013] Specifically, in step (2), PDP and BSA are dissolved in deionized water respectively, mixed in a mass ratio of (4:1) to (1:4), centrifuged at 10000 to 12000 rpm for 15 to 20 min to obtain PDP@BSA, and redispersed in deionized water; the concentration of the obtained PDP@BSA solution is 10 μg / mL to 40 mg / mL.

[0014] Specifically, in step (3), the cyclophosphamide aqueous solution is added dropwise to the PDP@BSA solution, and then the solution is shaken for 24 to 36 hours, and then centrifuged (12000 rpm, 10 to 20 minutes) to obtain a nanogel-like PDP@BSA / CYC complex.

[0015] Preferably, the mass concentration of the cyclophosphamide aqueous solution is 2-20 mg / mL; and the cyclophosphamide loading in the obtained PDP@BSA / CYC complex is 5%-30%.

[0016] Furthermore, the PAMAM dendrimer-loaded cyclophosphamide drug delivery system prepared by the above preparation method is also within the protection scope of the present invention.

[0017] Furthermore, the present invention also claims to protect the use of the above-mentioned PAMAM dendrimer-loaded cyclophosphamide drug delivery system in the preparation of drugs for treating systemic sclerosis. Beneficial Effects

[0018] (1) The nanogel-loaded cyclophosphamide delivery system formed by combining polyphenol-modified PAMAM with bovine serum albumin can combine the antioxidant properties of polyphenols with the various advantages of PAMAM, while introducing bovine serum albumin to reduce the cytotoxicity of the high-generation PAMAM strong cation, and jointly load cyclophosphamide to target the lesion site, effectively exerting antioxidant, anti-inflammatory and immune regulation effects.

[0019] (2) The nanogel delivery system prepared by the present invention has good biocompatibility, can target the negatively charged extracellular matrix, and efficiently remove reactive oxygen species, thereby effectively solving the problem of lack of specificity in the distribution of cyclophosphamide in the body, improving the bioavailability of cyclophosphamide, and being used for the first time in the treatment of systemic sclerosis, providing a promising treatment strategy for the study of fibrotic diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0021] Figure 1 The present invention is a flow chart of the preparation process of the nanogel PDP@BSA / CYC for delivering cyclophosphamide to treat systemic sclerosis.

[0022] Figure 2 (A) for G5 PAMAM, PDP 1 H-NMR spectrum, (B) is the Fourier transform infrared spectrum.

[0023] Figure 3 Zeta potential (A) and particle size statistics (B) of free BSA, PDP, PDP@BSA and PDP@BSA / CYC.

[0024] Figure 4 (A) is the particle size distribution diagram of PDP@BSA and PDP@BSA / CYC. The upper right corner is the TEM micrograph of PDP@BSA. (B) and (C) are the TEM images of PDP and BSA, respectively. The lower right corner is the comparison image of PDP@BSA. Scale bar = 50 nm.

[0025] Figure 5 Statistical results of cell survival rate (A) and hemolytic toxicity (B) of PDP@BSA nanogels with different concentrations.

[0026] Figure 6 Statistical results of scavenging hydroxyl radicals (A) and superoxide anions (B) by PDP@BSA nanogels at different concentrations.

[0027] Figure 7 The figures are comparative diagrams of the therapeutic effects of the present invention applied to systemic sclerosis mice; wherein (A) is the weight change of mice at important time points during modeling and treatment, (B) is the statistical result of the levels of pro-inflammatory and pro-fibrotic markers in serum after 40 days, and (C) is Masson staining of skin tissue, with a scale bar of 100 μm. DETAILED DESCRIPTION

[0028] The present invention can be better understood with reference to the following examples.

[0029] The experimental methods used in the following examples are conventional methods unless otherwise specified, and the reagents, methods and equipment used are conventional reagents, methods and equipment in the art unless otherwise specified. Example 1

[0030] A method for preparing a nanogel-loaded cyclophosphamide delivery system formed by combining catechol-modified PAMAM with bovine serum albumin, comprising the following steps: (1) Synthesis and characterization of PDP: 3,4-dihydroxybenzaldehyde and G5 PAMAM were dissolved in anhydrous methanol solution, mixed and reacted at 4°C for 24 hours, and anhydrous acetic acid was added as a catalyst. Then sodium cyanoborohydride was added and the reaction continued for 4 hours. After the reaction, the product was transferred to a 3500Da dialysis bag and dialyzed with deionized water to remove impurities. Finally, the purified light yellow powder was collected by vacuum freeze drying to obtain PDP; (2) Preparation of PDP@BSA solution: The PDP synthesized in step (1) was mixed with BSA and incubated at room temperature for 30 minutes, centrifuged (12000 rpm, 20 min) to obtain PDP@BSA, and redispersed in deionized water; (3) Loading cyclophosphamide: After the cyclophosphamide solution was added dropwise to the PDP@BSA solution, the mixture was shaken for 24 hours and centrifuged to obtain the PDP@BSA / CYC complex, i.e., the nanogel-loaded cyclophosphamide delivery system formed by the combination of catechol-modified PAMAM and bovine serum albumin.

[0031] The G5 PAMAM described in step (1) is a fifth-generation polyamide-amine dendrimer, and its surface group is amino group, with a molecular weight of 28826; the feeding molar ratio of 3,4-dihydroxybenzaldehyde to G5 PAMAM is 96:1; the feeding molar ratio of sodium cyanoborohydride to 3,4-dihydroxybenzaldehyde is 2:1.

[0032] The BSA in step (2) has a molecular weight of approximately 66.4 kDa, is composed of 583 amino acid residues, and has an isoelectric point of 4.7; PDP and BSA are respectively dissolved in deionized water, mixed in a mass ratio of 1:1, centrifuged (12000 rpm, 20 min) to obtain PDP@BSA, and redispersed in deionized water; the concentration of the PDP@BSA solution is 10 μg / mL to 40 mg / mL.

[0033] The cyclophosphamide solution was added dropwise to the PDP@BSA solution, and then shaken for 24 hours, and centrifuged (12000 rpm, 20 min) to obtain the PDP@BSA / CYC complex. Example 2

[0034] Synthesis and characterization of polyphenol-modified PAMAM: 92 mg of 3,4-dihydroxybenzaldehyde and 200 mg of G5 PAMAM were dissolved in anhydrous methanol solution, mixed and reacted at 4°C for 24 hours, and 200 microliters of anhydrous acetic acid was added as a catalyst. Then 83 mg of sodium cyanoborohydride was added and the reaction continued for 4 hours. After the reaction, the product was transferred to a 3500Da dialysis bag and dialyzed with deionized water to remove impurities. Finally, the purified light yellow powder was collected by vacuum freeze drying to obtain 153.6 mg of PDP; the product was used 1 H NMR (Bruker AscendTM 600MHz, Germany) and Fourier transform infrared spectroscopy (Thermo Fisher Scientific Nicolet iS20, USA) were used for characterization. Figure 2 .

[0035] Compared with G5 PAMAM, PDP 1 The H NMR spectrum showed additional chemical shifts at around 6.5-7.0 ppm and 4.0 ppm, corresponding to the aromatic and methylene protons of the catechol group, respectively. The Fourier transform infrared spectrum of PDP showed a 3408 cm -1 The absorption peaks at 1640 cm caused by the stretching vibration of the benzene ring are -1 The absorption peak is enhanced. Example 3

[0036] Preparation of PDP@BSA solution: 200 μg of PDP synthesized in step (1) was dissolved in water, mixed with 200 μg of BSA and incubated at room temperature for 30 minutes, then the liquid was added to 1 mL and centrifuged (12000 rpm, 20 min), and the precipitate was redispersed with 1 mL of deionized water; Zeta-sizer nanoparticle size potential instrument was used to measure Zeta potential and particle size distribution. The results are shown in FIG. Figure 3 and Figure 4 Compared with the negatively charged free BSA, the surface potential of PDP@BSA increased due to the binding of PDP (Figure 3A). When the mass ratio of PDP to BSA was 1:1, the hydrated particle size was 137.9 ± 7.66 nm, which increased to 257.0 ± 26.32 nm after the addition of cyclophosphamide ( Figure 3B). Transmission electron microscopy (TEM) directly captured the morphology of PDP@BSA, which is completely different from that of PDP and BSA ( Figure 4 ).

[0037] NIH 3T3 cells (Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd., Cat. No. ZM0096) were incubated in nanogels with different concentrations (0, 30, 90, 120, 180 μg / mL) for 24, 48, and 72 h, and the cell viability was detected using the CCK-8 kit. The results are shown in Figure 5 As shown in A, the survival rate of NIH 3T3 cells remained above 85%.

[0038] Take 500 μL of eyeball blood from BALB / c female mice and dilute it 10 times with PBS, and collect red blood cells by centrifugation (5000 rpm, 10 minutes). Repeat the washing and centrifugation with PBS until the supernatant is clear. After removing the supernatant, add 10 mLPBS to resuspend the precipitate. Incubate 200 μL of red blood cell suspension with 800 μLPBS, water or different concentrations of PDP@BSA at 37 °C for 4 hours. After centrifugation again, use a multifunctional microplate reader to detect the absorbance of the supernatant at 577 nm. Calculate the hemolysis rate ratio: Hemolysis rate (%) = [(absorbance 样品 − Absorbance 阴性 ) / (Absorbance 阳性 − Absorbance 阴性 )] × 100%; The results are as follows Figure 5 As shown in Figure 2B, within the evaluated concentration range (30-150 μg / mL), the hemolytic concentration of PDP@BSA remained below 5%, indicating that PDP@BSA has excellent blood compatibility.

[0039] The in vitro antioxidant capacity of different concentrations of PDP@BSA was determined using superoxide anion free radical and hydroxyl free radical assay kits.

[0040] The results are as follows Figure 6 As shown in the figure, PDP@BSA showed a dose-dependent clearance for both, and exhibited excellent antioxidant capacity at low doses. Example 4

[0041] Verification of the efficacy of PDP@BSA / CYC in systemic sclerosis: PDP@BSA / CYC was injected into SSc model mice through blood. The weight changes of mice were detected. After the treatment, the whole blood of mice was taken and placed at room temperature for 2 hours to precipitate the serum. The supernatant was taken by centrifugation (3000rpm, 20min), and the cytokines such as TGF-β, TNF-α and IL-1β were quantitatively analyzed by enzyme-linked immunosorbent assay (ELISA). The skin samples were subjected to histopathological analysis. The results are shown in Figure 7 As shown, mice treated with CYC or PDP@BSA / CYC gained weight, had decreased levels of TGF-β, TNF-α, and IL-1β in serum, thinned skin, and reduced fibrosis. The PDP@BSA / CYC treatment group had better efficacy, which was closer to normal levels.

[0042] The present invention provides a PAMAM dendrimer-based cyclophosphamide drug delivery system and its preparation method and application ideas and methods. There are many methods and ways to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A method for preparing a cyclophosphamide drug delivery system based on PAMAM dendrimers, characterized in that: The steps include: (1) Synthesis of polyphenol-modified PAMAM 3,4-dihydroxybenzaldehyde and G5 PAMAM were used as raw materials for reaction and then reduced with sodium cyanoborohydride to generate polyphenol-modified PAMAM, referred to as PDP; (2) Preparation of PDP@BSA solution The PDP synthesized in step (1) and BSA were mixed and incubated at room temperature for 20 to 40 minutes, centrifuged to obtain PDP@BSA, and redispersed in deionized water; (3) Loading cyclophosphamide The cyclophosphamide aqueous solution is added dropwise to the PDP@BSA solution synthesized in step (2) for coating, and the mixture is obtained by centrifugation.

2. The method for preparing the cyclophosphamide drug delivery system based on PAMAM dendrimers according to claim 1, characterized in that: In step (1), the G5 PAMAM is a fifth generation polyamide-amine dendrimer, and its surface group is amino group.

3. The method for preparing the cyclophosphamide-loaded drug delivery system based on PAMAM dendrimers according to claim 1, characterized in that: In step (1), the molar ratio of the 3,4-dihydroxybenzaldehyde to the G5 PAMAM is (24:1) to (128:1); the molar ratio of the sodium cyanoborohydride to the 3,4-dihydroxybenzaldehyde is (1.5:1) to (3:1).

4. The method for preparing the cyclophosphamide-loaded drug delivery system based on PAMAM dendrimers according to claim 1, characterized in that: In step (1), 3,4-dihydroxybenzaldehyde and G5 PAMAM are dissolved in an anhydrous methanol solution, mixed and reacted at 0-25° C. for 20-40 hours, and anhydrous acetic acid is added as a catalyst; sodium cyanoborohydride is then added to continue the reaction for 4-8 hours, and after the reaction, the product is fully dialyzed with deionized water to remove impurities, and finally the purified product is collected by vacuum freeze drying to obtain PDP.

5. The method for preparing the cyclophosphamide-loaded drug delivery system based on PAMAM dendrimers according to claim 1, characterized in that: In step (2), the number average molecular weight of BSA is 66-67 kDa.

6. The method for preparing the cyclophosphamide-loaded drug delivery system based on PAMAM dendrimers according to claim 1, characterized in that: In step (2), PDP and BSA are dissolved in deionized water respectively, mixed in a mass ratio of (4:1) to (1:4), centrifuged at 10000-12000 rpm for 15-20 min to obtain PDP@BSA, and redispersed in deionized water to obtain a PDP@BSA solution with a concentration of 10 μg / mL to 40 mg / mL.

7. The method for preparing the cyclophosphamide-loaded drug delivery system based on PAMAM dendrimers according to claim 1, characterized in that: In step (3), the cyclophosphamide aqueous solution is added dropwise to the PDP@BSA solution, and then shaken for 24 to 36 hours, and centrifuged (12000 rpm, 10 to 20 minutes) to obtain a PDP@BSA / CYC complex.

8. The method for preparing the cyclophosphamide-loaded drug delivery system based on PAMAM dendrimers according to claim 7, characterized in that: The mass concentration of the cyclophosphamide aqueous solution is 2-20 mg / mL; in the obtained PDP@BSA / CYC complex, the cyclophosphamide loading is 5-30 wt %.

9. The PAMAM dendrimer-loaded cyclophosphamide drug delivery system prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the PAMAM dendrimer-loaded cyclophosphamide drug delivery system according to claim 9 in the preparation of a drug for treating systemic sclerosis.

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